1. Field of the Invention
The present invention is related to a method for a (high through-put) screening detection of genetic modifications in engineering genome applied upon prokaryote and eukaryote cells or organism and a method for the monitoring of these genetic modifications through generations.
2. Description of the Related Art
In order to characterize the function of genetic sequences or for modulating the activity of genetic sequences, it is important to obtain introduction of genetic modifications into these sequences present in a cell genome. By the analysis of the defect (or advantage) induced by the genetic modification (the phenotype) the function of the gene can be identified.
This new approach has been developed in the field of molecular biology of prokaryote or eukaryote cells under the term “genome engineering”. This technique also applies for the production of genetically modified cells dedicated to genetic or cells therapy.
This field of research consists of a modification of specific sequences present in a cell genome (a prokaryote or an eukaryote cell genome). The introduced modification may result in inactivation of a gene expression, altered gene expression or modified gene expression and also modify gene sequence this is essential for studying genes function(s).
In addition, the same method can be used to correct a gene by restoring the normal phenotype as some therapeutics potential.
This approach also allows a person skilled in the art to generate from genetically modified cell lines (clones) individuals (prokaryote cells, eukaryote cells or organisms made of said cells) presenting a new physiological characteristic of an economic interest (such as prokaryote cells, eukaryote cells, plants or animals producing proteins or other compounds of interest).
Non human genetically engineered mammals (mouse, rat, sheep, etc) or plant models (Arabidopsis thaliana) are used for the study of human diseases or for improving plant or animal species.
Furthermore, animal models of human diseases are important to medical researches for studying the efficacy of new treatments before conducting clinical trials on human subjects.
This “genome engineering” method usually requires firstly an identification of a target genomic region in a cell genome, preferably a polynucleotide region encoding for a protein involved in a specific biochemical pathway.
Thereafter, the identified target polynucleotide region of the cell genome (hereafter called ‘target polynucleotide region’) can be genetically modified by gene targeting techniques that allow a substitution of at least one nucleotide with another nucleotide, a deletion of nucleotide sequences (of several Kb), or an incorporation of new nucleotide sequences into the cell genome.
These gene targeting techniques can be viewed as a form of artificial site directed in vivo mutagenesis.
These genetic modifications can be definitive (present in all the cells of an individual) or maintained for a specific delay under controlling elements (present in some specific cells of an individual under the control of the scientists performing the experiments) (M. Bunting et al. Genes & Dev. 1999, vol. 13, p. 1524-1526) and are preferably obtained by a recombination event that allows integration of exogenous nucleotide sequences in this ‘target polynucleotide region’ of the cell genome.
Typically, for this genetic modification an exogenous nucleotide sequence incorporated into a vector (insertion vector or replacement vector, such as a plasmid or a virus) will comprise a nucleotide fragment of interest flanked by two recombination arms.
These recombination arms share common sequence portions of homology with two flanking sequences of a target site of the target polynucleotide region.
Therefore, an homologous recombination event between the two recombination arm sequences and the flanking sequence of the target site will result in the insertion of the exogenous nucleotide sequence into the target site.
Advantageously, the target site of the target polynucleotide region could correspond to a gene which following the recombination event is therefore replaced by the exogenous nucleotide sequence.
The vector according to the invention or the exogenous nucleotide sequence may further comprise markers of positive or negative selection. Based upon the activity of these markers (markers of positive selection) or based upon the deletion of these markers (markers of negative selection) into specific cell lines (clones), it is possible to identify and select cells having integrated these exogenous nucleotide sequences. Positive selectable markers are preferably detectable by addition of antibiotics to cell cultures or are detected by light emission (US patent application 2004/0214222).
However, among these selected transformed cell lines, only a minority of the cells have integrated correctly the exogenous nucleotide sequences into the target polynucleotide region of the cell genome.
It is also necessary to perform additional screening step(s) upon each of these cell lines (these cells have a common ancestor and represent thus a clone) in order to identify and select which cell lines (which clone) have integrated the exogenous nucleotide sequences correctly (e.g. in the target polynucleotide region of the cell genome, in the correct orientation and with the number of required copies).
After this screening step(s), the clones of interest are recovered for a possible regeneration of complete individual(s) (poly-cellular eukaryotic organisms, such as an animal or a plant) from this recovered genetically modified cell line.
These screening steps are extremely long, costly, and require qualified people for maintaining all these cell lines alive (usually by a cryo-conservation of these cell lines).
Furthermore, a cryo-conservation reduces heavily the recovery cell lines and therefore affects the quality of the experience.
However, all the methods of the state of the art which are extremely long and complicated, cannot identify if the selected transformed cell lines comprise the number of required copies of foreign nucleotide sequences following multiple integration.
Indeed, this type of multiple integration with a high number of copies may present several drawbacks, especially in the field of plant genetics.
Homologous recombination in mammalian cells is a very rare occurrence. In embryonic totipotent mouse cells (ES cell) said occurrence is generally about 1% or lower depending on different factors.
The frequency of homologous recombination depends on the cell characteristics, the sequence homology degree between recombination arms and target sequences and the length of these recombination arms.
Gene targeting by homologous recombination has been achieved in some somatic mammalian cells, where the rate is usually lower than 1/1000. In plant cells, the homologous recombination degree is also very low.
Therefore, in order to improve the frequency of homologous recombination in these cells, it is usually necessary to either select some specific somatic cells of this animal or plant, which can be submitted to a higher percentage of homologous recombination or to improve the characteristics of a vector which comprises longer recombination arms.
However, the development of such vector is rather complicated and expensive.